Beta-Hexosaminidase is a key enzyme required for the lysosomal degradation of glycolipids, glycoproteins and glycosaminoglycans. Biosynthesis of active enzyme entails numerous posttranslational modifications of its two subunits, including association into dimers. Only the alpha-beta heterodimer (A isoenzyme) is able to hydrolyze GM2 ganglioside. Mutations in the genes encoding the alpha- and beta-subunits give rise to the catastrophic neurodegenerative disorders, Tay-Sachs disease and Sandhoff disease, respectively, as well as to GM2 gangliosidoses of later onset and milder course. Such mutations, already known to be very heterogeneous, will be characterized at the level of the genes and the proteins in order to understand the molecular basis of this class of diseases and to provide better diagnosis and carrier detection. Additional mutations will be engineered at specific sites of the alpha- and beta-subunit cDNAs to determine the effect of structural alterations on transport, dimerization and catalysis. The effect of premature translation termination on the metabolism of alpha-subunit cDNA will be examined because of its relevance to the major Tay-Sachs disease mutation. To remedy the current lack of knowledge of the three-dimensional structure of the enzyme, the disulfide bridges, which lock the subunits into their folded state, will be mapped and their biosynthetic pathway determined; an overexpression system will be developed to generate homogeneous enzyme for crystallographic studies. The goal is to fully understand structure/function relationships in the beta-hexosaminidase protein and their disruption in genetic disease.